Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that GM-CSF-differentiated macrophages are present in atherosclerotic plaques and promote lipid accumulation in aortas. However, it has not been determined if murine GM-CSF-differentiated macrophages take up LDL to become cholesterol-rich foam cells. In this study, murine bone marrow-derived cells were differentiated into macrophages with GM-CSF. GM-CSF-differentiated macrophages from LDL receptor-null mice incubated with native LDL showed massive, progressive cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of I-125 LDL also showed non-saturable macrophage LDL uptake that was linearly-related to the amount of LDL added, indicating that LDL uptake was receptor independent and mediated by fluid-phase pinocytosis. Since previous studies showed an important role for PI3K in atherosclerosis development and macrophage fluid-phase pinocytosis, we investigated the role of PI3K in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3K gamma inhibitor AS605240 showed a 50% reduction in LDL uptake and net cholesterol accumulation compared with macrophages incubated with LDL only. Incubation of PI3K gamma-null macrophages with LDL showed a similar 50% reduction in LDL uptake and net cholesterol accumulation compared with wild-type macrophages incubated with LDL. These results show that GM-CSF-differentiated murine macrophages become cholesterol-rich foam cells by fluid-phase pinocytosis of native LDL and identify PI3K gamma as contributing to this process. Our findings provide mechanistic insight for the decreased vascular lipid accumulation previously observed during atherosclerosis development in mice lacking PI3K gamma.